Immunomodulatory effects of colchicine on peripheral blood mononuclear cell subpopulations in human obesity: Data from a randomized controlled trial.

OBJECTIVE: Colchicine is known to reduce inflammation and improve endothelial cell function and atherosclerosis in obesity, but there is little knowledge of the specific circulating leukocyte populations that are modulated by colchicine. METHODS: A secondary analysis of a double-blind randomized controlled trial of colchicine 0.6 mg or placebo twice daily for 3 months on circulating leukocyte populations and regulation of the immune secretome in 35 adults with obesity was performed. RESULTS: Colchicine altered multiple innate immune cell populations, including dendritic cells and lymphoid progenitor cells, monocytes, and natural killer cells when compared with placebo. Among all subjects and within the colchicine group, changes in natural killer cells were significantly positively associated with reductions in biomarkers of inflammation, including cyclooxygenase 2, pulmonary surfactant-associated protein D, myeloperoxidase, proteinase 3, interleukin-16, and resistin. Changes in dendritic cells were positively correlated with changes in serum heart-type fatty acid-binding protein concentrations. Additionally, colchicine treatment reduced cluster of differentiation (CD) CD4+ T effector cells and CD8+ T cytotoxic cells. Conversely, colchicine increased CD4+ and CD8+ T central memory cells and activated CD38High CD8+ T cells. Changes in CD4+ T effector cells were associated with changes in serum heart-type fatty acid-binding protein. CONCLUSIONS: In adults with obesity, colchicine significantly affects circulating leukocyte populations involved in both innate and adaptive immune systems along with the associated inflammatory secretome.

Effects of colchicine on lipolysis and adipose tissue inflammation in adults with obesity and metabolic syndrome.

OBJECTIVE: The aim of this study was to examine whether colchicine's anti-inflammatory effects would improve measures of lipolysis and distribution of leukocyte populations in subcutaneous adipose tissue (SAT). METHODS: A secondary analysis was conducted for a double-blind, randomized, placebo-controlled pilot study in which 40 adults with obesity and metabolic syndrome (MetS) were randomized to colchicine 0.6 mg or placebo twice daily for 3 months. Non-insulin-suppressible (l0 ), insulin-suppressible (l2 ), and maximal (l0 +l2 ) lipolysis rates were calculated by minimal model analysis. Body composition was determined by dual-energy x-ray absorptiometry. SAT leukocyte populations were characterized by flow cytometry analysis from biopsied samples obtained before and after the intervention. RESULTS: Colchicine treatment significantly decreased l2 and l0 +l2 versus placebo (p < 0.05). These changes were associated with a significant reduction in markers of systemic inflammation, including high-sensitivity C-reactive protein, resistin, and circulating monocytes and neutrophils (p < 0.01). Colchicine did not significantly alter SAT leukocyte population distributions (p > 0.05). CONCLUSIONS: In adults with obesity and MetS, colchicine appears to improve insulin regulation of lipolysis and reduce markers of systemic inflammation independent of an effect on local leukocyte distributions in SAT. Further studies are needed to better understand the mechanisms by which colchicine affects adipose tissue metabolic pathways in adults with obesity and MetS.

Characterization of the adiponectin promoter + Cre recombinase insertion in the Tg(Adipoq-cre)1Evdr mouse by targeted locus amplification and droplet digital PCR.

The Tg(Adipoq-cre)1Evdr mouse has become an important tool in adipose tissue biology. However, the exact genomic transgene integration site has not been established. Using Targeted Locus Amplification (TLA) we found the transgene had integrated on mouse chromosome 9 between exons 6 and 7 of Tbx18. We detected transgene-transgene fusion; therefore, we used droplet digital polymerase chain reaction to identify Cre copy number. In two separate experiments, we digested with BAMHI and with HindIII to separate potentially conjoined Cre sequences. We found one copy of intact Cre present in each experiment, indicating transgene-transgene fusion in other parts of the BAC that would not contribute to tissue-specific Cre expression. Cre copy number for Tg(Adipoq-cre)1Evdr mice can be potentially used to identify homozygous mice.

Resistin mitigates stemness and metabolic profile of human adipose-derived mesenchymal stem cells via insulin resistance.

During obesity adipose tissue abundantly secrete pro-inflammatory adipokines like Tumour Necrosis factor-alpha (TNFα), resistin, leptin, etc. but reduced anti-inflammatory adipokines like adiponectin, interleukin (IL)-10, and IL-4. In our recent clinical study, it was observed that both gene expressions and stored levels of resistin were elevated in adipose tissue of metabolically obese Indians. Resistin profoundly increases obesity, mitigates lipid metabolism, and causes peripheral insulin resistance. It dysregulates the metabolism of human adipocytes but, its effects on human adipose-derived mesenchymal stem cells (hADSC) are sparsely explored. Therefore, the present study was designed to explore the repercussion of resistin on stemness and metabolic profile of hADSC. hADSC were isolated from a healthy individual followed by immunophenotyping. Purified cells were treated with resistin and proliferation was monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Cell Cycle experiments. Gene expressions of pluripotent markers, inflammatory mediators, and lipogenic genes were scrutinized. Insulin sensitivity was examined by western blot and glucose uptake assay. Further, consequences of resistin on differentiation potentials of hADSC were examined by temporal expressions of phospho (p)SMAD1/5/8 protein complex, non-phosphorylated beta (ß) catenin, and their dependent adipogenic transcription factors (ATF) and osteogenic transcription factors (OTF). MTT and cell cycle analysis revealed that resistin hampered proliferation of hADSC. Expressions of inflammatory markers and lipogenic genes were elevated. Resistin impaired insulin sensitivity and thus embarked insulin resistance in hADSC. Resistin increased adipogenesis and osteogenesis by altering expressions of activated pSMAD1/5/8 complex, activated ß catenin, ATF and OTF temporally. Downregulation of CCAAT/enhancer-binding proteins (C/EBP)α and adiponectin in adipocytes and Sirtuin (SIRT)1 in osteocytes denote that resistin induces immaturity and insulin resistance in adipocytes and osteocytes. This is the first study which, reports that resistin mitigates the stemness of hADSC by reducing proliferation, inducing insulin resistance, and hampering maturation of adipocyte and osteocyte which could lead to metabolic disorders.

Associations of GlycA and high-sensitivity C-reactive protein with measures of lipolysis in adults with obesity.

BACKGROUND: Obesity-associated inflammation promotes metabolic dysfunction. However, it is unclear how different inflammatory biomarkers predict dysregulation in specific tissues/organs, particularly adipose tissue. OBJECTIVE: The aim of our study was to examine whether GlycA, a nuclear magnetic resonance-measured biomarker of inflammation, is a better predictor of insulin-suppressible lipolysis and other measures of metabolic dysfunction compared with high-sensitivity C-reactive protein (hsCRP) in human obesity. METHODS: This was a cross-sectional study of 58 nondiabetic adults with obesity (body mass index: 39.8 ± 7.0 kg/m2, age 46.5 ± 12.2 years, 67.2% female) who underwent a frequently sampled intravenous glucose tolerance test in the fasted state. Noninsulin-suppressible (l0), insulin-suppressible (l2), and maximal (l0+l2) lipolysis rates, as well as insulin sensitivity and acute insulin response to glucose, were calculated by minimal model analysis. Nuclear magnetic resonance was used to measure GlycA. Body composition was determined by dual-energy X-ray absorptiometry. RESULTS: GlycA was strongly correlated with hsCRP (r = +0.46; P < .001). GlycA and hsCRP were positively associated with l2, l0+l2, and fat mass (Ps < .01). In linear regression models accounting for age, race, sex, and fat mass, GlycA remained significantly associated with l2 and l0+l2 (Ps < .05), whereas hsCRP did not (Ps ≥ .20). Neither GlycA nor hsCRP was associated with l0, insulin sensitivity, or acute insulin response to glucose. CONCLUSIONS: GlycA was associated with elevated lipolysis, independent of adiposity, in adults with obesity. Our findings suggest that GlycA and hsCRP have distinct inflammation-mediated metabolic effects, with GlycA having a greater association with adipose tissue dysfunction. Further studies are warranted to investigate the mechanisms underlying these associations.

Colchicine's effects on metabolic and inflammatory molecules in adults with obesity and metabolic syndrome: results from a pilot randomized controlled trial.

OBJECTIVE: Recent clinical trials have demonstrated that colchicine may have metabolic and cardiovascular and benefits in at-risk patients; however, the mechanisms through which colchicine may improve outcomes are still unclear. We sought to examine colchicine's effects on circulating inflammatory and metabolic molecules in adults with obesity and metabolic syndrome (MetS). METHODS: Blood samples were collected pre- and post-intervention during a double-blind randomized controlled trial in which 40 adults with obesity and MetS were randomized to colchicine 0.6 mg or placebo twice-daily for 3 months. Serum samples were analyzed for 1305 circulating factors using the SomaScan Platform. The Benjamini-Hochberg procedure was used to adjust the false discovery rate (FDR) for multiple testing. RESULTS: At baseline, age (48.0 ± 13.8 vs. 44.7 ± 10.3 years) and BMI (39.8 ± 6.4 vs. 41.8 ± 8.2 kg/m2) were not different between groups. After controlling for the FDR, 34 molecules were significantly changed by colchicine. Colchicine decreased concentrations of multiple inflammatory molecules, including C-reactive protein, interleukin 6, and resistin, in addition to vascular-related proteins (e.g., oxidized low-density lipoprotein receptor, phosphodiesterase 5A). Conversely, relative to placebo, colchicine significantly increased concentrations of eight molecules including secreted factors associated with metabolism and anti-thrombosis. CONCLUSIONS: In adults with obesity, colchicine significantly affected concentrations of proteins involved in the innate immune system, endothelial function and atherosclerosis, uncovering new mechanisms behind its cardiometabolic effects. Further research is warranted to investigate whether colchicine's IL-6 suppressive effects may be beneficial in COVID-19.

Influence of obese phenotype on metabolic profile, inflammatory mediators and stemness of hADSC in adipose tissue.

BACKGROUND: Unhealthy dietary practices, sedentary life style and lack of physical exercise in developing countries like India are major contributors of metabolic syndrome like obesity and diabetes. Obesity in Indians is defined at Body Mass Index (BMI, kg/m2) >25 and characterized as metabolically obese. OBJECTIVE: A preliminary study performed to explore ramification of obesity on metabolic profile of adipose tissue and adipose derived stem cells (ADSC) from control and obese Indians. METHODS: Adipose tissue/lipoaspirates from both control (BMI ≤ 23) subjects, and non-diabetic obese Indians subjects (BMI ≥ 25), were scrutinized for expressions of lipogenic genes, inflammatory mediators, stored adipokine levels, and insulin signaling proteins. Further, hADSC were isolated and immune-phenotyped from both the subject groups. Comparative assessments between chADSC and ohADSC were carried out for growth kinetics, expressions of pluripotent genes, adipogenic transcriptional factors, RUNX2, inflammatory mediators (IM), insulin signaling proteins, adipogenic and osteogenic differentiation. RESULTS: Adipose tissue of obese subjects depicted high leptin and resistin levels with reduced adiponectin levels. Expressions of IM and insulin signaling proteins were elevated compared to those of control subjects. hADSC of obese subjects demonstrated diminished proliferation, altered pluripotent genes, aggravated inflammation, adipogenesis with reduced osteogenesis. hADSC of obese had established insulin resistance compared to those of control subjects. CONCLUSION: This is the first study that describes hADSC of metabolically obese Indians have insulin resistance at lower BMI compared to Caucasians exemplifying plausible role in diminishing stemness of hADSC. Study alarms Indians to restore healthy dietary habits and assess quality of hADSC in regenerative therapy.

Swertiamarin ameliorates oleic acid induced lipid accumulation and oxidative stress by attenuating gluconeogenesis and lipogenesis in hepatic steatosis.

Swertiamarin, a bitter secoiridoid glycoside, is an antidiabetic drug with lipid lowering activity meliorates insulin resistance in Type 2 Diabetes condition. Therefore, the study was designed to explore the antioxidant and hypolipidemic activity of swertiamarin in ameliorating NAFLD caused due to hepatic lipid accumulation, inflammation and insulin resistance. Steatosis was induced in HepG2 cells by supplementing 1mM oleic acid (OA) for 24h which was marked by significant accumulation of lipid droplets. This was determined by Oil Red O (ORO) staining and triglyceride accumulation. Swertiamarin (25µg/ml) decreased triglyceride content by 2 folds and effectively reduced LDH release (50%) activity by protecting membrane integrity thus, preventing apoptosis evidenced by reduced cleavage of Caspase 3 and PARP1. We observed that swertiamarin significantly increased the expressions of major insulin signaling proteins like Insulin receptor (IR), PI(3)K, pAkt with concomitant reduction in p307 IRS-1. AMPK was activated by swertiamarin action, thus restoring insulin sensitivity in hepatocytes. In addition, qPCR results confirmed OA up-regulated Sterol Regulatory Element Binding Protein (SREBP)-1c and fatty acid synthase (FAS), resulting in increased fatty acid synthesis. Swertiamarin effectively modulated PPAR-α, a major potential regulator of carbohydrate metabolism which, in turn, decreased the levels of the gluconeogenic enzyme PEPCK, further restricting hepatic glucose production and fatty acid synthesis. Cumulatively, swertiamarin targets potential metabolic regulators AMPK and PPAR-α, through which it regulates hepatic glycemic burden, fat accumulation, insulin resistance and ROS in hepatic steatosis which emphasizes clinical significance of swertiamarin in regulating metabolism and as a suitable candidate for treating NAFLD.

Anti-apoptotic and cytoprotective effect of Enicostemma littorale against oxidative stress in Islets of Langerhans.

Context Oxidative stress induces apoptosis within Islets of Langerhans in diabetes mellitus (DM). Enicostemma littorale blume, herb of the Gentianaceae family is used as an anti-diabetic agent across rural India. Objective This report demonstrates potent anti-apoptotic and cyto-protective activity of Enicostemma littorale MeOH extract (EL MeOH ext.) against 50 µM H2O2 in isolated rat Islets. Materials and methods In this study, the whole plant methanolic extract of EL with doses 0.25-4 mg/mL each for the preincubation duration of 0.5-4 h against 50 µM H2O2 were tested for optimum protective dose and time by Trypan blue dye exclusion assay. Islet intracellular reactive oxygen species (ROS) was quantified by DCFDA staining and cell death using PS/PI & FDA/PI staining. Further, comet assay, biochemical assessment of caspase-3 and antioxidant enzyme activities along with immunoblotting of PARP-1, caspase-3, TNF-α activation and p-P38 MapK (stress kinase) induction was performed. Results The optimized dose of EL MeOH ext. 2 mg/mL for 2 h was used throughout the study, which significantly decreased total Intracellular ROS and cell death. Further, caspase-3 activity, PARP-1 cleavage, p-P38 MapK (stress kinase) activation and TNF-α levels, which had been significantly elevated, were normalized. Antioxidant enzymes like catalase, superoxide dismutase, reduced glutathione and glutathione peroxidase, along with Comet assay, demonstrated that pretreatment with EL MeOH ext. can augment antioxidant enzyme activities and protect from DNA damage. Discussion and conclusions Significant anti-apoptotic and cyto-protective effects were mediated by EL with Islets of Langerhans subjected to oxidative stress-induced cell death.

Insulin resistance: an additional risk factor in the pathogenesis of cardiovascular disease in type 2 diabetes.

Sedentary life style and high calorie dietary habits are prominent leading cause of metabolic syndrome in modern world. Obesity plays a central role in occurrence of various diseases like hyperinsulinemia, hyperglycemia and hyperlipidemia, which lead to insulin resistance and metabolic derangements like cardiovascular diseases (CVDs) mediated by oxidative stress. The mortality rate due to CVDs is on the rise in developing countries. Insulin resistance (IR) leads to micro or macro angiopathy, peripheral arterial dysfunction, hampered blood flow, hypertension, as well as the cardiomyocyte and the endothelial cell dysfunctions, thus increasing risk factors for coronary artery blockage, stroke and heart failure suggesting that there is a strong association between IR and CVDs. The plausible linkages between these two pathophysiological conditions are altered levels of insulin signaling proteins such as IR-ß, IRS-1, PI3K, Akt, Glut4 and PGC-1α that hamper insulin-mediated glucose uptake as well as other functions of insulin in the cardiomyocytes and the endothelial cells of the heart. Reduced AMPK, PFK-2 and elevated levels of NADP(H)-dependent oxidases produced by activated M1 macrophages of the adipose tissue and elevated levels of circulating angiotensin are also cause of CVD in diabetes mellitus condition. Insulin sensitizers, angiotensin blockers, superoxide scavengers are used as therapeutics in the amelioration of CVD. It evidently becomes important to unravel the mechanisms of the association between IR and CVDs in order to formulate novel efficient drugs to treat patients suffering from insulin resistance-mediated cardiovascular diseases. The possible associations between insulin resistance and cardiovascular diseases are reviewed here.

Swertiamarin: An Active Lead from Enicostemma littorale Regulates Hepatic and Adipose Tissue Gene Expression by Targeting PPAR- γ and Improves Insulin Sensitivity in Experimental NIDDM Rat Model.

Enicostemma littorale (EL) Blume is one of the herbs widely used for treating and alleviating the effects of both type I and type II diabetes. However, lack of understanding of mechanism precludes the use of the herb and its molecules. In this study, we attempt to unravel the molecular mechanism of action of swertiamarin, a compound isolated form EL, by comparing its molecular effects with those of aqueous EL extract in alleviating the insulin resistance in type II diabetes. We further investigated hypolipidemic and insulin sensitizing effect of swertiamarin in experimentally induced noninsulin dependent diabetes mellitus (NIDDM) in rats. Swertiamarin (50 mg/kg) and aqueous extract (15 grams dried plant equivalent extract/kg) were administered to rats orally for 40 days and tight regulation of serum glucose, insulin, and lipid profile was found in both groups. Their mode of action was by restoring G6Pase and HMG-CoA reductase activities to normal levels and restoring normal transcriptional levels of PEPCK, GK, Glut 2, PPAR- γ , leptin, adiponectin, LPL, SREBP-1c, and Glut 4 genes. This suggests that both treatments increased insulin sensitivity and regulated carbohydrate and fat metabolism. This is the first report on the role of SM in regulating the PPAR γ -mediated regulation of candidate genes involved in metabolism in peripheral tissues in vivo.